PROJECT SUMMARY Aedes aegypti females have evolved efficient metabolic pathways for managing the high ammonia concentrations that are released during a blood meal's digestion. Carbon (C) atoms from glucose are required for clearance of ammonia and excess nitrogen (N) disposal through the interplay of multiple pathways, including glycolysis, and ammonia fixation, assimilation and excretion pathways. What remains unknown is how these intersecting metabolic pathways are regulated. The long-term goal is to identify the biochemical and molecular bases underlying the regulation of N and C metabolism in Ae. aegypti, so that novel metabolism- based strategies for mosquito control can be developed as a way to improve public health and quality of life. The overall objective for this application is to identify the mechanisms involved in the regulation of polyamines and glucose/ammonia metabolism. The central hypothesis is that the proper disposition of N waste is controlled by uric acid and polyamine fluxes, and by proteins involved in the last step of glycolysis. The rationale for the proposed research is that the identification of regulatory mechanisms will provide new opportunities for the subsequent identification of targets for the design of innovative strategies to mosquito control. Guided by strong preliminary data, the central hypothesis will be tested by pursuing two specific aims: 1) Determine the metabolic flux of polyamines and the mechanisms of its regulation in blood-fed mosquitoes; 2) Identify mechanisms of regulation of both glucose and ammonia metabolism in mosquitoes. Under the first aim, stable isotopically labeled compounds and advanced LC/MS methods, western blots, qRT-PCR, chemical inhibitors and reverse genetics techniques will be used. In the second aim, standard techniques for gene cloning, protein purification, kinetic characterization, crystallization and structure determination will be performed. Further, immunoprecipitation, inmunofluorence, RNA interference, and metabolomics analysis will be performed. This approach is innovative because it combines classical and state-of-the-art techniques (i) to monitor metabolite flux at atomic level without sample derivatization and (ii) to identify regulatory mechanisms of polyamine, glucose/ammonia metabolism at different levels including transcriptional and post-translational levels. The proposed research is significant because it is expected to fill gaps in current understanding of how female mosquitoes maintain N and C metabolism and regulate the proper disposition of N waste upon a blood meal without which ammonia levels could reach lethal concentrations. Thus, these results are expected to uncover mosquito-specific regulatory mechanisms under high demands of ammonia detoxification. As such, a much-improved fundamental understanding of the biochemical and molecular bases underlying the N and C metabolism in mosquitoes can be anticipated. It is also expected that what can be learned in Ae. aegypti mosquitoes through traditional and advanced technologies will be broadly applicable to identify regulatory mechanisms in other arthropod vectors of diseases, as well as in other biological systems.